The biotransformation of steroids by conjugation to highly charged chemical groups such as glucosiduronidate and sulfonate is a process of immense importance that results in the conversion of these essentially hydrophobic compounds to a more water soluble state. While the glucosiduronidation of steroids is considered preparatory to their ultimate excretion, the biological import of steroid sulfonation remains a poorly understood. Sulfonation, the transfer of a sulfonate group from a universal donor to an appropriate acceptor site, is carried out by enzymes termed sulfotransferases. There are multiple biochemical and functional implications of steroid sulfonation. For instance, by increasing steroid polarity and water solubility, sulfonation can influence steroid transportability or function as a steroid trapping and storage mechanism. By acting as either an initiating event (steroid sulfonate is the active form) or a terminating event (steroid sulfonate is the inactive form), sulfonation can function to regulate steroid physiologic responsivity. An interesting and challenging conundrum is the fact that one of the most active tissues to sulfonate steroids is the adrenal cortex. Thus, in an effort to shed light on the biological consequences of steroid sulfonation in the mammalian adrenal cortex, the SSR is engaged in the isolation, cloning and transcriptional regulation of adrenocortical steroid sulfotransferases. Three steroid sulfotransferases, namely estrogen sulfotransferase (EST) and two stereoselective 3-hydrosysteroid sulfotransferases (3a-HST/3b-HST) are the current focus. Molecular cloning and expression of EST and 3a-HST have been completed; the cloning of 3b-HST is in progress. The structural basis for the exquisite stereospecificity of the HSTs will be examined by sequence analysis, mutational studies and chimeric constructs. The 5'- flanking region of the EST gene has been cloned and the complete structural gene is now being determined. EST is a unique protein of multiple charge isoforms that demonstrate distinct catalytic and high affinity steroid binding activities. It is of particular interest that EST has a nuclear localization and that EST and the HSTs are differentially expressed in functionally distinct adrenocortical zones. The binding site for the activated sulfonate donor 3'-phosphoadenosine-5'- phosphosulfate (PAPS) has been identified and the precise amino acid residues involved in the PAPS-protein interaction have been determined by mutational studies. Enzymes responsible for the synthesis of PAPS are being purified and cloned. Additionally, a PAPS-specific sulfatase is undergoing purification for biochemical analysis and cloning.